Craniofacial skeletal muscle defects occur in congenital deformities, such as hemifacial microsomia and facial/palatal clefts: in certain myopathies, such as muscular dystrophy; and as a result of surgical procedures for oral cancer or trauma. Recent advances have been made in reporting soft-tissue contour in patients with craniofacial anomalies, including hemifacial microsomia and clefts of lip and palate. Success in the repair or replacement muscle defects is limited by difficulty in muscle transplantation and survival. A potential solution would be to develop tissue engineering protocols in which myoblasts are directly injected to augment existing muscle fibers or, alternatively, in which skeletal muscle could be generated in vitro from harvested satellite myoblasts and implanted in the defective site. Such protocols would require isolation of muscle satellite cells, their proliferation and differentiation to myofibers, and, importantly, formation of the myotendinous junction (MTJ) that transduces force generated by muscle to its connective tissue attachment site. Another important application is the introduction of myoblasts for cell-based gene therapy. The long-term objective of the proposed studies is to devise methods for isolating of skeletal muscle satellite cells, to optimize their culture and conversion to myofibers, and to characterize the formation of the MTJ in vitro. Expression of the alpha7beta1 integrin is developmentally regulated and is tissue-specific for skeletal muscle and its precursor satellite cells. Expression of the alpha7beta1 integrin is developmentally regulated and is tissue-specific for skeletal muscle and its precursor satellite ells. We have shown that the alpha7 integrin is not only expressed on satellite cells but is also concentrated at the MTJ in terminally differentiated myotubes. We propose to examine the function of the alpha7 receptor in mediating MTJ formation and to use this receptor as a tissue-specific marker for the isolation of skeletal muscle satellite cells. Our specific aims are: 1) Test whether the function of alpha7 integrin is regulated by beta1D subunit partner during formation of the MTJ. 2) Design tissue-engineered 3-D skeletal muscle myofiber organoids derived from sorted satellite cells. 3) Clone the human alpha7 integrin cDNA and generate specific antibodies for the isolation for human satellite ells. These studies will enhance understanding of the assembly of the MTJ and suggest new approaches in tissue engineering to promote reconstruction/augmentation of craniofacial skeletal muscle defects caused by disease, trauma, or surgical procedures.